Generation and analysis of mammalian cells containing various Raf mutations

The generation of a raf-1 allele where the gene is flanked by two loxP sites ('floxed') in ES cells was described with the aim to enable the conditional deletion of raf-1 in mice generated from the mutated Es cells. Two separate targeting vectors were designed that each harboured a loxP site flanked by homologous raf-1 sequences, with one targeted to the 5' end of raf-1 and the other targeted to the 3' end of raf-1. These vectors were used for gene targeting and eight ES cell clones were identified that had undergone homologous recombination with both targeting vectors. These ES clones were used to generate chimaeric mice and germline transmission was achieved from one of the targeted ES cell clones. A Cre expression plasmid was used to successfully mediate the deletion of the raf-1 floxed gene in ES cells.;The role of A-Raf and Raf-1 in cell growth and survival was investigated by using primary mouse embryonic fibroblasts that either lacked A-Raf, Raf-1, or that contained a mutation of the two endogenous tyrosine residues at 340/341 of Raf-1 to phenylalanine residues (RafFF) which result in undetectable kinase activity towards MEK. No differences were observed with growth and proliferation of the A-Raf deficient, Raf-1 deficient or RafFF MEFs as compared with wild-type MEFs. An increased susceptibility of the Raf-1 deficient MEFs to undergo apoptosis was observed upon treatment with etoposide and upon treatment with an anti-CD95 antibody plus cycloheximide. However, the RafFF MEFs did not exhibit the same susceptibility to undergo apoptosis. These results suggest that Raf-1 has a role in cell survival that is independent from its role in the Ras/Raf/MEK/ERK cascade.